Browsing by Subject "Ejaculate fractions"

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    Aldose reductase B1 in pig seminal plasma: identification, localization in reproductive tissues, and relationship with quality and sperm preservation
    (Frontiers Media, 2021-06-08) Mateo Otero, Yentel; Viñolas Vergés, Estel; Llavanera, Marc; Ribas Maynou, Jordi; Roca, Jordi; Yeste, Marc; Barranco, Isabel; Medicina y Cirugía Animal
    Aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme that belongs to the aldo-keto reductase protein superfamily, has been reported to be involved in both male and female reproductive physiology. The objectives of this study were: (1) to evaluate the concentration of SP-AKR1B1 in pig ejaculate fractions; (2) to describe the immunohistochemical localization of AKR1B1 alongside the boar genital tract; (3) to evaluate the relationship between SP-AKR1B1 and sperm quality/functionality parameters. Ejaculates from seven boars (one ejaculate per boar) were collected in separate portions [the first 10 mL of the sperm rich fraction (SRF-P1), the rest of the SRF (SRF-P2), and the post-SRF (PSRF)], and the concentration of SP-AKR1B1 was assessed using an enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry and immunoblotting targeting was conducted in the reproductive tissues of these boars. Additionally, the entire ejaculates of 14 boars (one ejaculate per boar) were collected and split into three separate aliquots for: (i) SP-AKR1B1 quantification; (ii) assessment of sperm concentration and morphology; and (iii) evaluation of sperm quality and functionality parameters upon ejaculate collection (0 h) and after 72 h of liquid storage at 17°C. Concentration of AKR1B1 in the SP of SRF-P1 (458.2 ± 116.33 ng/mL) was lower (P < 0.05) than that of SRF-P2 (1105.0 ± 229.80 ng/mL) and PSRF (1342.4 ± 260.18 ng/mL). Monomeric and dimeric AKR1B1 forms were expressed alongside the reproductive tissues, except in the bulbourethral glands. No relationship between SP-AKR1B1 and sperm quality/functionality parameters was observed either at 0 h or after 72 h of storage at 17°C. In conclusion, AKR1B1 is expressed in the reproductive organs of boars (except bulbourethral glands) and a higher concentration is found in the PSRF suggesting that seminal vesicles would be the main secretory source. However, this enzyme does not appear to be related to sperm quality/functionality or to the sperm ability to withstand liquid storage at 17°C.
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    Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction
    (BMC, 2023-11-06) Viñolas Vergés, Estel; Ribas Maynou, Jordi; Barranco, Isabel; Peres Rubio, Camila; Bonet, Sergi; Roca, Jordi; Yeste, Marc; Medicina y Cirugía Animal
    Background protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated. Results This study determined chromatin protamination (Chromomycin A3 test, CMA3), condensation (Dibromobi‑mane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the frst 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the diferent ejaculate fractions (P>0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF (P=0.018 and P=0.004, respectively). Regarding DNA integrity, no diferences between fractions were observed (P>0.05). As the SRF-P1 has the highest sperm concentra‑tion and ejaculate fractions are known to difer in antioxidant composition, the oxidative stress index (OSi) in SP, calcu‑ lated as total oxidant activity divided by total antioxidant capacity, was tested and confrmed to be higher in the SRFP1 than in SRF-P2 and PSRF (0.42±0.06 vs. 0.23±0.09 and 0.08±0.00, respectively; P<0.01); this index, in addition, was observed to be correlated to the sperm concentration of each fraction (Rs=0.973; P<0.001). Conclusion While sperm DNA integrity was not found to difer between ejaculate fractions, SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF. This could be related to the OSi of each fraction.
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    Cryogenic electron microscopy reveals morphologically distinct subtypes of extracellular vesicles among porcine ejaculate fractions
    (Nature Research, 2024-07-13) Parra, Ana; Barranco, Isabel; Martínez Díaz, Pablo; González, Esperanza; Albóniga, Oihane E.; Cabrera, Diana; Falcón Pérez, Juan M.; Roca, Jordi; Medicina y Cirugía Animal
    Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.
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    Seminal plasma antioxidants are directly involved in boar sperm cryotolerance
    (Elsevier, 2018-02) Li, Junwei; Barranco Cascales, Isabel; Tvarijonaviciute, Asta; Molina, Manuel F.; Martínez García, Emilio; Rodríguez Martínez, Heriberto; Parrilla Riera, Inmaculada; Roca Aleu, Jorge; Medicina y Cirugía Animal
    Boar ejaculates are ejected in fractions with a specific composition in terms of sperm numbers and seminal plasma (SP), which is reflected in the varying sperm cryotolerance observed among different fractions. As boar sperm are particularly sensitive to oxidative stress, this study evaluated the role of SP antioxidants in the observed differences in sperm cryotolerance among ejaculate fractions. Ten ejaculates from five boars were manually collected in fractions: the first 10 mL of the sperm-rich fraction (SRF), the rest of the SRF and the post-SRF. Semen samples comprising the entire ejaculate (EE) were created by proportionally mixing the three fractions described above. Each of the 40 resulting semen samples was split into two aliquots: one was used for sperm cryopreservation following a standard protocol utilizing 0.5-mL straws, and the other was used to collect SP for antioxidant assessment. Frozen-thawed (FT) sperm from the SRF (the first 10 mL of the SRF and the rest of the SRF) and those from post-SRF were of the highest and worst quality, respectively, which was measured in terms of total and objective progressive motility and viability (P < 0.01). Viable FT sperm from the post-SRF generated more reactive oxygen species and experienced more lipid peroxidation than those from the SRF (both the first 10 mL and the rest of the SRF) (P < 0.01). The percentage of FT sperm exhibiting fragmented nuclear DNA did not differ among ejaculate fractions and the EE. Catalase, glutathione peroxidase and glutathione peroxidase 5 (GPx-5) were lowest in SP from the first 10 mL of the SRF (P < 0.001), whereas superoxide dismutase (SOD) and paraoxonase 1 (PON-1) were highest in SP of the SRF (both the first 10 mL and the rest of the SRF) (P < 0.01). Trolox-equivalent antioxidant capacity (TEAC) and the ferric-reducing ability of plasma (FRAP) were highest in SP from the first 10 mL of the SRF and lowest in the post-SRF (P < 0.001), whereas cupric-reducing antioxidant capacity was lowest (P < 0.05) in SP from the first 10 mL of the SRF. Regression analyses indicated that certain SP antioxidants had good predictive value for post-thaw recovery rates of total motility (R2 = 54.8%, P < 0.001; including SOD, TEAC and FRAP) and viability (R2 = 56.1%, P < 0.001; including SOD, PON-1, GPx-5 and TEAC). These results demonstrated that certain SP antioxidants are positively involved in boar sperm cryotolerance, minimizing the oxidative stress imposed by cryogenic handling.
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    The seminal plasma of the boar is rich in cytokines, with significant individual and intra-ejaculate variation
    (Wiley, 2015-12) Barranco Cascales, Isabel; Rubér, Marie; Pérez Patiño, Cristina; Atikuzzaman, Mohammad; Martínez García, Emilio; Roca Aleu, Jorge; Rodríguez Martínez, Heriberto; Medicina y Cirugía Animal
    Problem: The boar, as human, sequentially ejaculates sperm-rich and sperm-poor fractions. Seminal plasma (SP) spermadhesins (PSP-I/PSP-II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility. Methods: SP samples from the sperm-rich fraction (SRF) and the sperm-poor post-SRF fraction (post-SRF) of manually collected ejaculates from eight boars (four ejaculates per boar) were analysed by commercial multiplex bead assay kits (Milliplex MAP, Millipore, USA) for interferon-γ, interferon gamma-induced protein 10, macrophage-derived chemokine, growth-regulated oncogene, granulocyte-macrophage colony-stimulating factor, monocyte chemo-attractant protein-1, interleukins (IL)-6, IL-8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-β3. Results: Cytokine concentrations differed between the ejaculate fractions among boars, being highest in the post-SRF. Conclusion: Boar SP is rich in Th1, Th2, Th17 and Th3 cytokines, with lowest concentrations in the sperm-peak-containing fraction, indicating its main immune influence might reside in the larger, protein-rich sperm-poor post-SRF.